To examine whether BRCA and PTEN alteration status affect the anti-tumor efficacy of olaparib/MPD3 combination, we performedin vivoexperiments on Hs578t-tumor and BT549-tumor bearing balb/cnude mice. As expected, olaparib monotherapy in Hs578t or BT549 tumors did not show detectable tumor suppressing efficacy, which could be ascribed to the poor sensitivity of olaparib in BRCA wild-type tumors. Interestingly, Hs578t tumors showed delayed onset of tumor growth inhibition effect by MPD3 monotherapy, producing 48.4% TGI rate on the last day of observation. Following an unexpected response to MPD3 in PTEN wild-type tumors, different control tumor specimens at day 1, 16, and 28 were examined by immunohistochemical staining to investigate tumor microenvironmental changes during the drug treatment course. We found evidence of increasing pattern of active caspase-3 expression over the course of time, which is thought to be associated with activation of localized extracellular prodrug and late tumor inhibiting efficacy in Hs578t tumors. Owing to the poor response to olaparib therapy, both MPD3 and olaparib/MPD3 displayed similar tumor inhibiting efficacies in Hs578t tumor bearing mice, suggesting that olaparib triggered apoptosis is needed to generate synergistic efficacy with MPD3. Lastly,in vivoexperiment results with BT549 tumor xenografted model confirmed the contribution of PTEN-loss alteration to thein vivoactivity of MPD3. MPD3 monotherapy was able to induce substantial tumor growth inhibition with 63.4% TGI. We ascribe these results mainly to the PTEN-loss induced upregulation of the macropinocytosis level, witnessed by significantly increased albumin uptake level in BT549cells. However, as with Hs578t xenograft model, the addition of olaparib did not cause significant tumor growth inhibition. Note that despite the comparable anti-cancer activity of olaparib/docetaxel to that of olaparib/MPD3, docetaxel combination treatment caused severebody weight loss, with 60% and 20% lethality in Hs578t and in BT549 tumor bearing mice, respectively. The results ofin vivoanti-cancer efficacies of MPD3 and olaparib in TNBC tumor models expressing different biomarkers are summarized inTable 1. Coefficients of drug interaction (CDI) values of the combination of olaparib/MPD3 were 0.04, 1.22 and 0.42 in MDA-MB-436, Hs578t, BT549 xenograft models, respectively, where CDI of <1, =1, or >1 indicates that the combination therapy is synergistic, additive, or antagonistic. These results provide compelling evidence that the BRCA and PTEN alteration status influence the efficacy of our combination strategy, showing that BRCA mutation and PTEN-loss contribute to the favorable anti-cancer efficacy of the strategy.

To examine whether BRCA and PTEN alteration status affect the anti-tumor efficacy of olaparib/MPD3 combination, we performedin vivoexperiments on Hs578t-tumor and BT549-tumor bearing balb/cnude mice. As expected, olaparib monotherapy in Hs578t or BT549 tumors did not show detectable tumor suppressing efficacy, which could be ascribed to the poor sensitivity of olaparib in BRCA wild-type tumors. Interestingly, Hs578t tumors showed delayed onset of tumor growth inhibition effect by MPD3 monotherapy, producing 48.4% TGI rate on the last day of observation. Following an unexpected response to MPD3 in PTEN wild-type tumors, different control tumor specimens at day 1, 16, and 28 were examined by immunohistochemical staining to investigate tumor microenvironmental changes during the drug treatment course. We found evidence of increasing pattern of active caspase-3 expression over the course of time, which is thought to be associated with activation of localized extracellular prodrug and late tumor inhibiting efficacy in Hs578t tumors. Owing to the poor response to olaparib therapy, both MPD3 and olaparib/MPD3 displayed similar tumor inhibiting efficacies in Hs578t tumor bearing mice, suggesting that olaparib triggered apoptosis is needed to generate synergistic efficacy with MPD3. Lastly,in vivoexperiment results with BT549 tumor xenografted model confirmed the contribution of PTEN-loss alteration to thein vivoactivity of MPD3. MPD3 monotherapy was able to induce substantial tumor growth inhibition with 63.4% TGI. We ascribe these results mainly to the PTEN-loss induced upregulation of the macropinocytosis level, witnessed by significantly increased albumin uptake level in BT549cells. However, as with Hs578t xenograft model, the addition of olaparib did not cause significant tumor growth inhibition. Note that despite the comparable anti-cancer activity of olaparib/docetaxel to that of olaparib/MPD3, docetaxel combination treatment caused severebody weight loss, with 60% and 20% lethality in Hs578t and in BT549 tumor bearing mice, respectively. The results ofin vivoanti-cancer efficacies of MPD3 and olaparib in TNBC tumor models expressing different biomarkers are summarized inTable 1. Coefficients of drug interaction (CDI) values of the combination of olaparib/MPD3 were 0.04, 1.22 and 0.42 in MDA-MB-436, Hs578t, BT549 xenograft models, respectively, where CDI of <1, =1, or >1 indicates that the combination therapy is synergistic, additive, or antagonistic. These results provide compelling evidence that the BRCA and PTEN alteration status influence the efficacy of our combination strategy, showing that BRCA mutation and PTEN-loss contribute to the favorable anti-cancer efficacy of the strategy.

To examine whether BRCA and PTEN alteration status affect the anti-tumor efficacy of olaparib/MPD3 combination, we performedin vivoexperiments on Hs578t-tumor and BT549-tumor bearing balb/cnude mice. As expected, olaparib monotherapy in Hs578t or BT549 tumors did not show detectable tumor suppressing efficacy, which could be ascribed to the poor sensitivity of olaparib in BRCA wild-type tumors. Interestingly, Hs578t tumors showed delayed onset of tumor growth inhibition effect by MPD3 monotherapy, producing 48.4% TGI rate on the last day of observation. Following an unexpected response to MPD3 in PTEN wild-type tumors, different control tumor specimens at day 1, 16, and 28 were examined by immunohistochemical staining to investigate tumor microenvironmental changes during the drug treatment course. We found evidence of increasing pattern of active caspase-3 expression over the course of time, which is thought to be associated with activation of localized extracellular prodrug and late tumor inhibiting efficacy in Hs578t tumors. Owing to the poor response to olaparib therapy, both MPD3 and olaparib/MPD3 displayed similar tumor inhibiting efficacies in Hs578t tumor bearing mice, suggesting that olaparib triggered apoptosis is needed to generate synergistic efficacy with MPD3. Lastly,in vivoexperiment results with BT549 tumor xenografted model confirmed the contribution of PTEN-loss alteration to thein vivoactivity of MPD3. MPD3 monotherapy was able to induce substantial tumor growth inhibition with 63.4% TGI. We ascribe these results mainly to the PTEN-loss induced upregulation of the macropinocytosis level, witnessed by significantly increased albumin uptake level in BT549cells. However, as with Hs578t xenograft model, the addition of olaparib did not cause significant tumor growth inhibition. Note that despite the comparable anti-cancer activity of olaparib/docetaxel to that of olaparib/MPD3, docetaxel combination treatment caused severebody weight loss, with 60% and 20% lethality in Hs578t and in BT549 tumor bearing mice, respectively. The results ofin vivoanti-cancer efficacies of MPD3 and olaparib in TNBC tumor models expressing different biomarkers are summarized inTable 1. Coefficients of drug interaction (CDI) values of the combination of olaparib/MPD3 were 0.04, 1.22 and 0.42 in MDA-MB-436, Hs578t, BT549 xenograft models, respectively, where CDI of <1, =1, or >1 indicates that the combination therapy is synergistic, additive, or antagonistic. These results provide compelling evidence that the BRCA and PTEN alteration status influence the efficacy of our combination strategy, showing that BRCA mutation and PTEN-loss contribute to the favorable anti-cancer efficacy of the strategy.